We tested the hypothesis that the mechanisms involved in the more rapid onset of fatigue when O(2) availability is reduced in contracting skeletal muscle are similar to those when O(2) availability is more sufficient. Two series of experiments were performed in isolated, single skeletal muscle fibers from Xenopus laevis. First, relative force and free cytosolic Ca(2+) concentrations ([Ca(2+)](c)) were measured simultaneously in single fibers (n = 6) stimulated at increasing frequencies (0.25, 0.33, 0.5, and 1 Hz) at an extracellular PO(2) of either 22 or 159 Torr. Muscle fatigue (force = 50% of initial peak tension) occurred significantly sooner (P < 0.05) during the low- (237 +/- 40 s) vs. high-PO(2) treatments (280 +/- 38 s). Relative [Ca(2+)](c) was significantly decreased from maximal values at the fatigue time point during both the high- (72 +/- 4%) and low-PO(2) conditions (78 +/- 4%), but no significant difference was observed between the treatments. In the second series of experiments, using the same stimulation regime as the first, fibers (n = 6) exposed to 5 mM caffeine immediately after fatigue demonstrated an immediate but incomplete relative force recovery during both the low- (89 +/- 4%) and high-PO(2) treatments (82 +/- 3%), with no significant difference between treatments. Additionally, there was no significant difference in relative [Ca(2+)](c) between the high- (100 +/- 12% of prefatigue values) and low-PO(2) treatments (108 +/- 12%) on application of caffeine. These results suggest that in isolated, single skeletal muscle fibers, the earlier onset of fatigue that occurred during the low-extracellular PO(2) condition was modulated through similar pathways as the fatigue process during the high and involved a decrease in relative peak [Ca(2+)](c).